Histone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid Improves Energetic Status and Cardiomyogenic Differentiation of Human Dilated Myocardium-Derived Primary Mesenchymal Cells

Figures

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  Figure 1. The phenotype and proliferation rate of healthy and pathological human myocardium-derived mesenchymal stromal cells (hmMSC). (A) Obtaining of hmMSC from the human heart explants after five days of cultivation, scale bar = 500 μm. Morphology of healthy (B) and pathological (C) hmMSC, scale bar = 640 μm. (D) Graphical representation of attachment area for healthy and pathological cell. The distribution of 60 cells of each type according to the attached area are presented. (E) Expression of surface markers on healthy and pathological hmMSC. The proliferation rate of healthy and pathological hmMSC evaluated by metabolic Cell Counting Kit-8 (CCK8) (F) and cell-counting method (G). Data are shown as mean ± standard deviation (SD). The * p ≤ 0.05, n = 6 from three experiments calculated by an Excel program. Total adherent surface of different cell types and the minor and major axes of healthy and pathological cells are presented as Supplementary Figure S1.

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  Figure 2. Energetic status of healthy and pathological hmMSC. (A) Mitochondrial membrane potential measured by 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC1) dye. (B) Level of ATP in healthy and pathological hmMSCs (picomoles (pM) of adenosine triphosphate (ATP) per cell). Representative micrographs of electron microscope of healthy (C) and pathological (D) hmMSC are shown, scale bar = 2 µm. Yellow arrows indicate mitochondria. Data are shown as mean ± standard deviation (SD). The * p ≤ 0.05, ** p ≤ 0.01, n = 5 from three experiments. Student t test was calculated by Graphpad Prism 6 program.

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  Figure 3. Mitochondrial respiration and glycolytic capacity of healthy and pathological primary hmMSC. (A) Schematic representation of oxygen consumption and glycolytic acidification assays. (B) Mitochondrial activity of healthy and pathological hmMSC. (C) Glycolytic capacity of healthy and pathological hmMSC. Oxygen consumption (pM) and glycolytic acidification (milli pH (mpH)/min) values were normalized to protein level (mg/mL). Data are shown as mean ± standard deviation (SD). The * p ≤ 0.05, n = 3 from two experiments. Student t test was calculated by an Excel program.

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  Figure 4. HDAC activity and gene expression profile in healthy and pathological hmMSC. (A) HDAC activity in healthy and pathological hmMSC (arbitrary fluorescence units/per protein (mg/mL), n = 5. (B) Relative expression (2−ΔCT) of HDAC1–7 genes in healthy and pathological hmMSC, n = 3. Changed HDAC activity (C) and HDAC1–7 gene expression (2−ΔΔCT) (D) in healthy and pathological cells after exposure to 1 µM of SAHA for three days, n = 3. Gene expression was analyzed as follows: expression = 2−ΔCt × 100,000, where ΔCt = (Cttarget gene − Ctreference gene); relative expression = 2−ΔΔCt, where ΔΔCt is a subtraction of ΔCt of control cells from ΔCt of cells treated with SAHA. Data are shown as mean ± standard deviation (SD). The * p ≤ 0.05, ** p ≤ 0.01, n = 3–5 from 3 experiments. Student t-test was calculated by an Excel and Graphpad Prism 6 programs.

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  Figure 5. SAHA enhances the energetic status of healthy and pathological hmMSC. Mitochondrial membrane potential (A) and ATP production (B) in healthy and pathological hmMSCs exposed to 1 µM of SAHA for three days. Changes of oxygen consumption rate (OCR) ((pM)/protein (mg/mL)) in healthy (C) and pathological (D) cells and calculated mitochondrial parameters (E) after exposure to SAHA. Data are displayed as fold of changes with and without SAHA. Control–cells of both types not affected by SAHA. Data are shown as mean ± standard deviation (SD). The * p ≤ 0.05, n = 3 from 3 experiments. Student t-test was calculated by an Excel program.

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  Figure 6. The effect of SAHA on glycolysis of healthy and pathological hmMSC. Decrease of extracellular acidification rate (ECAR) ((mpH/min)/protein (mg/m)) in healthy cells (A) and pathological (B) cells after exposure to 1 µM of SAHA for three days. (C) Quantification of glycolytic stress parameters in healthy and pathological hmMSC with and without SAHA. Data are shown as mean ± standard deviation (SD). The * p ≤ 0.05, n = 3 from three experiments. Student t-test was calculated by an Excel program.

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  Figure 7. The impact of SAHA on the levels of transcription factor transcription factor NK2 Homeobox 5 (Nkx2.5), cardiac troponin T (TNNT2) and alpha cardiac actin (ACTC1), in healthy and pathological hmMSC. (A) Expression of Nkx2.5 gene (2−ΔΔCT) in healthy and pathological cells during 3–21 days of cultivation with 1 µM of SAHA. (B) Expression of TNNT2 gene (2−ΔΔCT) in healthy and pathological cells during 3–21 days of cultivation with 1 µM of SAHA. (C) Expression of ACTC1 gene (2−ΔΔCT) in healthy and pathological cells during 3–21 days of cultivation with 1 µM of SAHA. (D) Level of alpha cardiac actin protein compared to beta actin in healthy and pathological cells after exposure to 1 µM of SAHA analyzed by Western blotting. (E) Immunocytochemical micrographs of alpha cardiac actin levels in healthy and pathological cells after exposure to 1 µM of SAHA for 3–14 days, scale bar = 20 μm. Control-healthy and pathological cells without SAHA. Gene expression was analyzed as follows: expression = 2−ΔCt × 100,000, where ΔCt = (Cttarget gene − Ctreference gene); relative expression = 2−ΔΔCt, where ΔΔCt is a subtraction of ΔCt of control cells from ΔCt of cells treated with SAHA. Data are shown…